Nanopore electrode, nanopore membrane, methods of preparation and surface modification, and use thereof

ABSTRACT

Provided are fabrication, characterization and application of a nanodisk electrode, a nanopore electrode and a nanopore membrane. These three nanostructures share common fabrication steps. In one embodiment, the fabrication of a disk electrode involves sealing a sharpened internal signal transduction element (“ISTE”) into a substrate, followed by polishing of the substrate until a nanometer-sized disk of the ISTE is exposed. The fabrication of a nanopore electrode is accomplished by etching the nanodisk electrode to create a pore in the substrate, with the remaining ISTE comprising the pore base. Complete removal of the ISTE yields a nanopore membrane, in which a conical shaped pore is embedded in a thin membrane of the substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application represents a divisional application of U.S. applicationSer. No. 11/744,154, filed May 3, 2007 now U.S. Pat. No. 7,849,581 whichclaims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 60/919,659, filed Mar. 23, 2007 and U.S. ProvisionalApplication No. 60/797,850, filed May 5, 2006, the entirety of each ofwhich is incorporated by this reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant#FA9550-06-C-0006 awarded by the Defense Advance Research ProjectsAgency. This invention was also made with government support under grant#ES013548 awarded by the National Institutes of Health. This inventionwas also made with government support under grant CHE-0616505 awarded bythe National Science Foundation. The US government has certain rights tothis invention.

TECHNICAL FIELD

The invention relates to the field of nanotechnology. In particular, theinvention is related to nanodisk electrodes, nanopore electrodes andnanopore membranes.

BACKGROUND

Molecular transport in individual pores (e.g., protein ion channels ((a)Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. Proc. Natl.Acad. Sci. U.S.A. 1996, 93, 13770; (b) Bayley, H; Cremer, P. S. Nature2001, 413, 226; (c) Gu, L.-Q.; Braha, O.; Conlan, S.; Cheley, S. andBayley, H. Nature 1999, 398, 686) and synthetic channels ((a) Ito, T.;Sun, L.; Crooks, R. M. Anal. Chem. 2003, 75, 2399; (b) Ito, T.; Sun. L.;Henriquez, R. R.; Crooks, R. M. Acc. Chem. Res. 2004, 37, 937; (c)Hinds, B. J.; Chopra, N.; Rantell, T.; Andrews, R.; Gavalas, V.; Bachas,L. G. Science 2004, 303, 62. (d) Majumber, M.; Chopra, N.; Hings, B. J.J. Am. Chem. Soc. 2005, 127, 9062; (e) Li, J.; Gershow, M.; Stein, D.;Brandin, D.; Golovchenko, J. A. Nat. Mater. 2003, 2, 611; (f) Li, J.;Stein, D.; McMullan, C.; Branton, D.; Aziz, M. J.; Golovchenko, J. A.Nature 2001, 412, 166.)) and in materials containing pores of nanometerdimensions (e.g., zeolite catalysts and skin) are of interest throughoutchemistry and biology. It is generally recognized that transportselectivity, based on a chemical or physical property of the permeant,is often observed in pores when the size of the pore is sufficientlysmall that interactions between the pore surface and permeant influencelocal transport dynamics (“permeant” refers to a molecule or ion thatpasses through the pore). The rate of alkali metal ion transport throughgramicidin channels, for instance, is highly dependent on metal ionradius, a consequence of the channel radius (˜2 Å) being comparable tothe dehydrated ion radius ((a) Andersen, O. S.; Feldberg, S. W. J. Phys.Chem. 1996, 100, 4622; (b) Andersen, O. S. Biophys. J. 1983, 41, 147;(c) Andersen, O. S. Biophys. J. 1983, 41, 135). Longer rangeinteractions over a few to tens of nanometers (e.g., electrostaticforces) between the pore surface and permeant can also lead to transportselectivity in pores of larger dimensions ((a) Daiguji, H.; Yang, P.;Majumdar, A. Nano Lett. 2004, 4, 137; (b) Karnik, R.; Fan, R.; Yue, M.;Li, D.; Yang, P.'; Majumdar, A. Nano Lett., 2005, 5, 943).

Developments over the past several decades in understanding poretransport mechanisms and the origins of transport selectivity have ledto recent interest in the development of chemical and biological sensorsbased on selective transport through nanometer scale channels and pores.Protein ion channels, such as α-hemolysin, engineered or chemicallymodified to interact with a target analyte, are capable of detectingindividual molecules by measuring the modulation of ionic currentthrough the protein upon analyte binding (Meller, A. J. Phys. Condens.Matter 2003, 15, R581). The ability to observe molecule or particletransport dynamics within individual nanopores, rather than ensembledaveraged results, has motivated fundamental research on pores employingbiological as well as synthetic affinity pairs (Umezawa, Y.; Aoki, H.Anal. Chem. 2004, 76, 320 A).

In addition to biological pores, there have been significant advances inanalytic detection employing synthetic pores in recent years, madelargely possible by the rapid developments in methods and materials fornanoscale synthesis ((a) Jirage, K. B.; Hulteen, J. C.; Martin, C. R.Science 1997, 278, 655; (b) Harrell, C. C.; Lee, S. B.; Martin, C. R.Anal. Chem. 2003, 75, 6861 (c) Harrell, C. C.; Kohli, P. Siwy, Z.;Martin, C. R. J. Am. Chem. Soc. 2004, 126, 15646. (d) Fologea, D.;Gershow, M.; Ledden, B.; McNabb, D. S.; Golovchenko, J. A.; Li, JialiNano Lett. 2005, 5, 1905; (e) Fologea, D.; Gershow, M.; Uplinger, J;Thomas, B.; McNabb, D. S.; Li, Jiali Nano Lett. 2005, 5, 1734; (f) Chen,P.; Gu, J.; Brandin, E., Kin, Y.-R., Wang, Q.; Branton, D. Nano Lett.,2004, 4, 2293; (g) Storm, A. J.; Chen, J. H.; Ling, x. S.; Zandbergen,H. W.; Dekker, C. Nat. Mater. 2003, 2, 537; (h) Liu, N.; Dunphy, D. R.;Atanassov, P.; Bunge, S. D.; Chen, Z.; López, G. P.; Boyle, T. J.;Brinker, C. J. Nano Lett. 2004, 4, 551; (i) Fan, R. Karnik, R.; Yue, M.Li, D., Majumdar, A; Yang, P. Nano Lett. 2005, 5, 1633). For example,polycarbonate membranes that contain nanosize channels have beenemployed for the template synthesis of gold nanotubes, which can besubsequently functionalized for biosensor applications including thedetection of DNA molecules (Heins, E. A.; Siwy, Z. S.; Baker, L. A.;Martin, C. R. Nano Lett., 2005, 5, 1824.). pH-switchable ion transportselectivity has been achieved by attachment of cysteine at the surfaceof the Au nanotubes (Lee, S. B.; Martin, C. R. Anal. Chem. 2001, 73,768). Solid-state nanopores fabricated in Si₃N₄ membranes ((a) Fologea,D.; Gershow, M.; Ledden, B.; McNabb, D. S.; Golovchenko, J. A.; Li,Jiali Nano Lett. 2005, 5, 1905; (b) Fologea, D.; Gershow, M.; Uplinger,J; Thomas, B.; McNabb, D. S.; Li, Jiali Nano Lett. 2005, 5, 1734; (c)Chen, P.; Gu, J.; Brandin, E., Kin, Y.-R., Wang, Q.; Branton, D. NanoLett., 2004, 4, 2293; (d) Storm, A. J.; Chen, J. H.; Ling, x. S.;Zandbergen, H. W.; Dekker, C. Nat. Mater. 2003, 2, 537; (e) Liu, N.;Dunphy, D. R.; Atanassov, P.; Bunge, S. D.; Chen, Z.; Lopez, G. P.;Boyle, T. J.; Brinker, C. J. Nano Lett. 2004, 4, 551) have been used forsingle molecule analysis and DNA detection, and silicon nanotubes havebeen integrated with microfluidic systems for DNA sensing (Fan, R.Karnik, R.; Yue, M. Li, D., Majumdar, A; Yang, P. Nano Lett. 2005, 5,1633.) Carbon nanotubes (CNTs) have been employed as a nanoparticleCoulter counter (Ito, T.; Sun, L.; Crooks, R. M. Anal. Chem. 2003, 75,2399). Aligned and chemically modified CNTs, incorporated into polymerfilms to created multichannel membrane structures, are also capable ofreporting analyte binding (Majumber, M.; Chopra, N.; Hings, B. J. J. Am.Chem. Soc. 2005, 127, 9062).

The use of biological nanopores, for detection of single molecules hasbeen in practice for two decades (see, e.g., Deamer, D. W., Branton, D.,Acc. Chem. Res. 2002, 35, 817-825). For example, the biological proteinnanopore α-hemolysin (αHL) from Staphylococcus aureus has proven to beideal for single molecule detection, given the inner pore constrictiondiameter of 1.6 nm (Song, S., Hobaugh, M. R., Shustak, C., Cheley, S.,Bayley, H., Govaux, J. E., Science, 1996, 274, 1859-1865).

The use of nanometer-scale electrodes has also attracted considerableinterest as tools in fundamental research since the late 1980s. Forexample, nanoelectrodes have been employed in studies of fastelectron-transfer reactions (Watkins, J. J.; Chen, J.; White, H. S.;Abruña, H. D.; Maisonhaute, E.; and Amatore, C. Anal. Chem. 2003, 75,3962; Penner, R. M.; Heben, M. J.; Longin, T. L.; Lewis, N. S. Science1990, 250, 1118), interfacial structure (Conyers, J. L. Jr.; White, H.S. Anal. Chem. 2000, 72, 4441; Chen, S.; Kucemak, A. J. Phys. Chem. B2002, 106, 9396), single electron and single molecule electrochemistry(Fan, F-R. F.; Bard, A. J.; Science 1995, 267, 871; Fan, F-R, F.; Kwak,J.; Bard, A. J. J. Am. Chem. Soc. 1996, 118, 9669), as mimics of fuelcell catalysts (Chen, S.; Kucemak, A. J. Phys. Chem. B 2004, 108,13984), and as analytical probes in bioelectrochemical measurements(Wightman, R. M. Science 2006, 311, 1570).

Methods of fabricating nanometer-sized electrodes can be found inseveral reports (Zoski, C. G. Electroanalysis 2002, 14, 1041; Watkins,J. J.; Zhang, B.; White, H. S. J. Chem. Edu. 2005, 82, 712; Arrigan, D.W. M. Analyst 2004, 129, 1157). Most frequently, the end of anelectrochemically etched carbon fiber or metal wire is sealed into aninsulating material (e.g., glass, wax, and polymers) leaving the tip ofthe fiber or wire exposed (Penner, R. M.; Heben, M. J.; Lewis, N. S.Anal. Chem. 1989, 61, 1630; Huang, W-H.; Pang, D-W.; Tong, H.; Wang,Z-L.; Cheng, J-K. Anal. Chem. 2001, 73, 1048; Hrapovic, S.; Luong, J. H.T. Anal. Chem. 2003, 75, 3308; Slevin, C. J.; Gray, N. J.; Macpherson,J. V.; Webb, M. A.; Unwin, P. R. Electrochem. Comm. 1999, 1, 282; Woo,D-H.; Kang, H.; Park, S-M. Anal. Chem. 2003, 75, 6732). Electrodesfabricated in this way generally have a hemispherical or conical shapeshrouded by a thin layer of insulating material. The nature of theinsulator can restrict the use of the electrode. For example, electrodesinsulated with thin organic layers are simple to prepare, but their useis generally restricted to aqueous solutions, and they tend to exhibitprohibitively large capacitive currents in transient measurements due tothe capacitance of the thin insulating layer (Watkins, J. J.; Chen, J.;White, H. S.; Abruña, H. D.; Maisonhaute, E.; and Amatore, C. Anal.Chem. 2003, 75, 3962).

Nanometer sized disk electrodes have been fabricated by pulling Pt wiresembedded in glass capillaries with micro-pipette pullers andsubsequently exposing a disk-shaped area of the metal using mechanicalpolishes or chemical etchants (Ballesteros Katemann, B.; Schuhmann, W.Electroanalysis 2002, 14, 22). The resulting glass-shrouded electrodesare durable and have favorable electrical properties. However, usingthis procedure, it is difficult to prepare electrodes with consistentsizes. Moreover, the use of costly pipette pullers is required. AlthoughShao et al. mention the monitoring of resistance during the polishing ofglass-sealed Pt nano-electrodes (Shao, Y.; Mirkin, M. V.; Fish, G.;Kokotov, S.; Palanker, D.; Lewis, A. Anal. Chem. 1997, 69, 1627), nodetails of the methodology and instrumentation have been published.

SUMMARY OF INVENTION

Nanodisk Electrode

Provided is a nanodisk electrode, exemplified in FIG. 1(A), comprising asubstrate having a first surface and a second surface and an internalsignal transduction element (“ISTE”) having a first surface and a secondsurface. The ISTE is embedded in the substrate such that the firstsurface of the ISTE is within the same plane of the first surface of thesubstrate, and the second surface of the ISTE is extended, or exposedthrough the second surface of the substrate. The first surface of theISTE is defined as a “disk”, a “nanodisk” or “microdisk”, depending onthe radius of the exposed surface; and is exposed through the firstsurface of the ISTE. The substrate may be configured to include morethan one ISTE, or include an array of ISTEs.

In various embodiment, the substrate may be made of, for example, glass,Si, SiO₂, Si₃N₄, quartz, alumina, nitrides, metals, polymers or othersuitable materials. The substrate can be of a pure substance or acomposite. In particular embodiments, the substrate is a soda lime orlead glass capillary.

The ISTE may be of various suitable shapes. The ISTE may be made of anymaterial that is suitable for electrical signal transduction. The ISTEis preferably metal, such as, for example, platinum (“Pt”), gold (“Au”),silver (“Ag”), or tungsten (“W”) but may be any conducting material, forinstance carbon (“C”), a semiconductor (e.g., silicon, “Si”) orelectronically conducting polymer (e.g., polyanaline). In a particularembodiment, the ISTE comprises a platinum wire. The platinum wire may befurther attached to a tungsten rod via silver paint for externalelectrical connection to electronic instruments. The platinum wire mayalso be attached to other materials such as copper. In anotherparticular embodiment, the ISTE comprises an Au wire.

Further provided are methods of preparing a nanodisk electrode. Suchmethods comprises sealing a part comprising the ISTE in a substrate, andpolishing the substrate until the first surface, i.e., disk, of the ISTEis exposed.

Further provided are methods of preparing a nanodisk electrode with adisk of a desired radius. Such methods comprise providing an ISTE with aconical tip, sealing the conical tip of the ISTE in a substrate with apart comprising the second surface of the ISTE extended outside of thesubstrate, polishing the substrate using a polishing means in order toexpose the tip, measuring the electrical continuity resistance betweenthe ISTE and the polishing means, and stopping the polishing when themeasured resistance reaches a desired resistance. For example, duringthe polishing, an intermittent electrical measurement using a high-inputimpedance metal-oxide semiconductor field effect transistor(MOSFET)-based circuit is made to determine the resistance between theextended part of the ISTE and the polishing cloth. The polishing isimmediately stopped when the measured resistance meets a desiredresistance, which signifies the exposure of a disk of desired size. Thedesired resistance signifying the exposure of a disk of desirable sizemay be obtained empirically or determined by finite element simulationsand calibration curves.

Nanopore Electrode

Further provided is a nanopore electrode, as exemplified in FIG. 1(B),The nanopore electrode comprises a substrate having a first surface anda second surface, an ISTE having a first surface and a second surface,and a nanopore having an orifice, a base and an interior surface. TheISTE is embedded in the substrate such that the first surface of theISTE is the base of the nanopore, and the second surface of the ISTE isextended, or exposed through the second surface of the substrate. Theorifice of the nanopore opens through the first surface of thesubstrate. The interior surface of the nanopore is an integral part ofthe substrate. The first surface of the substrate may also be called theexterior surface of the nanopore. The substrate may be configured toinclude more than one nanopore and more than one ISTE, or include anarray of nanopores and ISTEs. A nanopore electrode can be incorporatedinto silicon and other microelectronic lithogaphically fabricateddevices.

In various embodiments, the substrate may be made of, for example,glass, Si, SiO₂, Si₃N₄, quartz, alumina, nitrides, metals, polymers orother suitable materials. The membrane can be of a pure substance or acomposite. In particular embodiments, the substrate is a soda lime orlead glass capillary.

The nanopore may assume various suitable shapes, preferably a truncatedcone shape with the radius of the orifice smaller than that of the baseof the nanopore. The radius of the orifice of a conical nanoporepreferably ranges from about 2 nm to about 500 nm, or larger. The radiusof the base ranges from 100 nm to the diameter of the wire used for theISTE. The depth of a conical nanopore is the distance from the orificeto the base of the nanopore. The depth is preferably ranging from 10 nmto 100 μm.

The ISTE may be of various suitable shapes. The ISTE may be made of anymaterial that is suitable for electrical signal transduction. The ISTEis preferably metal, such as, for example, Pt, Au, Ag, or W, but may beany conducting material, for instance C, a semiconductor (e.g., Si) orelectronically conducting polymer (e.g., polyanaline). In a particularembodiment, the ISTE comprises a platinum wire. The platinum wire may befurther attached to a tungsten rod via silver paint. The platinum wiremay also be attached to other materials such as copper. In anotherparticular embodiment, the ISTE comprises an Au wire.

The interior surface and/or the exterior surface of the nanopore may bemodified to change the surface properties, for example, the electricalcharge density, hydrophobicity or hydrophilicity, of the respectivesurfaces. The exterior surface of the nanopore may be modified by afirst entity. The interior surface of the nanopore may be modified by asecond entity. The first and second entities may be different entities.The first or second entities may be polymers, small organic molecules,proteins, etc. The modification of the surfaces may be physical orchemical in nature. For example, the first or second entity may beattached to the respective surfaces via noncovalent forces, e.g., byhydrophobic interactions. For another example, the first or secondentity may be attached to the respective surfaces via covalent bonds.The second entity may comprise chemical functionalities, e.g.,chemically reactive amino groups, or comprise functional binding sites,e.g., streptavidin attached to the interior surface providing biotinbinding sites. Alternatively, various functional sensor molecules may befurther attached, either by physical force, by chemical bonding or bycoordinate covalent bonds, to the second entities that are attached tothe interior surface of the nanopore to impart various functions to thenanopore. Lipid bilayers may be deposited across the orifice by variousmeans to serve as supports for proteins, enzymes and other biologicalmolecules that might serve as sensor transduction agents for interactingwith, detecting, and analysis of target analytes.

In certain embodiments, the exterior surface of a nanopore is chemicallymodified by an entity with a single chemical functionality. For example,a chemically reactive silane with an inert terminus, e.g.,Cl(Me)₂Si(CH₂)₃CN, is reacted to the exterior surface of a glassnanopore to generate a silane monolayer terminating in —CN groups. Otherreactive silanes with different terminus groups, and/or with differentsurface reactive groups (e.g., methoxy groups or multiple chlorinegroups), can similarly be attached to the surface to form monolayer andmultilayer films. The interior surface of a nanopore may be modifiedwith an entity with a single or multiple functionalities. For example,the interior of a glass nanopore may be silanized by EtO(Me)₂Si(CH₂)₃NH₂to yield a monolayer terminating in —NH₂ groups. Various functionalmolecules may be attached to the interior surface of the nanopore viareaction with the —NH₂ groups attached to the interior surface. Forexample, carboxylate groups of a sensor protein may react with the —NH₂groups and thus the protein is covalently attached to the interiorsurface of the nanopore via amide bonds. Alternatively, the interiorsurface may be directly modified with an entity with a functionalbinding site. For example, EtO(Me)₂Si(CH₂)₃NH-streptavidin can beattached to the interior surface of a glass nanopore thus imparting abiotin-binding property to the interior surface. Alternatively, theinterior surface of a nanopore may be modified by an entity comprising abait element, for instance, glutathione, such that another functionalentity that recognizes the bait element, for example, a sensor moleculewith a GST-tag (glutathione S-transferase tag), can be immobilized tothe interior surface of the nanopore by non-covalent bonds.

Further provided is a method of preparing a nanopore electrode, themethod comprising preparing a nanodisk electrode as disclosed herein,and etching the exposed surface of the ISTE to produce a nanopore in thesubstrate.

It is to be noted that the shape and the size of the part of the ISTEthat is sealed in the substrate partly defines the shape, and the sizeof the base and the orifice of the nanopore. For example, if the part ofthe ISTE that is sealed in the substrate is cylindrical, the resultingnanopore will be of a cylindrical shape. If the part of the ISTE that issealed in the substrate is conical, the resulting nanopore will be of atruncated conical shape.

Further provided are methods of preparing a chemically modified glassnanopore electrode. Such a method comprises providing a glass nanodiskelectrode as disclosed herein; modifying the first surface of the glassnanodisk electrode with a first entity; etching the exposed nanodisk toproduce a nanopore; and modifying the interior surface of the nanoporewith a second entity. The first surface of the glass nanodisk is alsothe exterior surface of the nanopore. In certain embodiments, theexterior surface of the nanopore is chemically modified withCl(Me)₂Si(CH₂)₃CN. The modification generates a silane monolayerterminating in —CN groups that protect the exterior surface from furtherreaction with other chemically reactive entities. One purpose of themodification of the exterior surface is to prevent modification of theexterior surface by an entity that modifies the interior surface of thenanopore, the exterior surface may be modified or coated with anyappropriate chemically inert species. After the nanopore is created, theinterior glass surface of the nanopore may be silanized by anEtO(Me)₂Si(CH₂)₃NH₂ to yield a monolayer terminating in a —NH₂ group.Various functional molecules can be attached to the interior surface ofthe nanopore via reaction with the —NH₂ groups attached to the interiorsurface. Other reactive silanes with different terminus groups, and/orwith different surface reactive groups (e.g., methoxy groups or multiplechlorine groups), can similarly be attached to the surface to formmonolayer and multilayer films. The interior surface of a nanopore maybe modified with an entity with a single or multiple functionalities.

Further provided are methods of forming a surface-modified nanoporeelectrode, the method comprising: providing a substrate having a firstsurface and a second surface wherein the first surface is modified by afirst entity to change the surface property of the first surface;providing an ISTE having a first surface and a second surface, whereinthe first surface of the ISTE is sealed in the substrate and the secondsurface of the ISTE extends, or exposed through the second surface ofthe substrate; providing a nanopore having an orifice opening throughthe first surface of the substrate, having a base wherein the base isthe first surface of the ISTE, and having an interior surface whereinthe interior surface is modified by a second entity to change thesurface property of the interior surface; and optionally providing afunctional entity attached to the interior surface of the nanopore viathe second entity.

The mass transport of a charged species through a nanopore can beelectrostatically gated “on” and “off” by controlling the electricalcharge density at the orifice of the nanopore. Accordingly, furtherprovided are methods of using a surface-modified nanopore electrode tocontrol the transport of a charged species. Such a method comprises:providing a sample solution containing at least one charged species tobe analyzed; providing a nanopore electrode including an ISTE, whereinthe interior surface of the nanopore is modified with a entity such thatthe electrical charge density at the pore orifice is adjustable by anappropriate adjusting species; contacting the nanopore electrode withthe solution such that the exterior surface of the nanopore is immersedin the solution and the nanopore is filled with the solution; applyingan appropriate voltage between the solution and the ISTE; add theappropriate adjusting species to the solution such that the electricalcharge density at the orifice is varied and that the at least onecharged species passing through the nanopore can be electrostaticallygated “on” and “off” by controlling the electrical charge density at theorifice; monitoring the electrical conductivity of the nanopore; andanalyzing the electrical conductivity to determine to what extent thetransfer of the charged species is controlled. The solution need notcontain a supporting electrolyte.

For example, the interior surface of a glass nanopore electrode ismodified by an entity with terminal —NH₂ groups. Adjusting thesolution's pH results in a reversible protonation of the —NH₂ groupsbound to the interior surface of the pore. Thus, the electrical chargedensity at the pore orifice may be controlled by varying the extent ofthe protonation of the —NH₂ groups on the interior surface of the pore.Accordingly, a protonation of the interior area of the nanoporeresulting in terminal —NH₃ ⁺ groups may prevent the entry of a positivecharged species into the nanopore due to electrostatic repulsion betweenthe —NH₃ ⁺ groups and the positive charged groups.

The surface-modified glass nanopore electrode can also be used tocontrol the rate of a redox reaction by adjusting the pH value of asample solution. For example, in a redox reaction, positive chargedspecies Rox is reduced to species Rred. Rred may be charged oruncharged. The pH value of the solution may be adjusted such that theprotonation of the interior area of the nanopore resulting in terminal—NH₃ ⁺ groups may prevent the positive charged species Rox from enteringinto the nanopore due to electrostatic repulsion between the —NH₃ ⁺groups and Rox. Accordingly, the rate of the reduction reaction from Roxto Rred is controlled by controlling the transport rate of Rox.

Also provided are methods of monitoring the pH of a solution. Such amethod comprises providing a sample solution containing a chargedspecies as a pH-indicating species; providing a glass nanopore electrodeincluding an ISTE, wherein the interior surface of the nanopore ismodified with a entity such that the electrical charge density at thepore orifice varies depending on the pH value of the solution;contacting the nanopore electrode with the solution such that theexterior surface of the nanopore is immersed in the solution and thenanopore is filled with the solution; applying an appropriate voltagebetween the solution and the ISTE; monitoring the electricalconductivity of the nanopore; and analyzing the electrical conductivityto determine to the pH of the solution.

A surface-modified nanopore electrode with functional entities attachedto the interior surface of the nanopore can be used as a sensor fordetection of chemical and biological molecules by measuring the changein the conductance of the pore upon binding of the analyte to thefunctional entity that is attached to the interior surface of thenanopore. Accordingly, provided is a method of using a nanoporeelectrode to monitor selective binding of analytes. The methodcomprises: providing a sample solution containing an analyte ofinterest; providing a nanopore electrode including an ISTE wherein theinterior surface of the nanopore is modified with a functional entitysuch that the functional entity selectively binds to the analyte ofinterest; contacting the nanopore electrode with the solution such thatthe exterior surface of the nanopore is immersed in the solution and thenanopore is filled with the solution; applying an appropriate voltagebetween the solution and the ISTE of the nanopore electrode; monitoringthe electrical conductivity of the nanopore; and analyzing theelectrical conductivity to determine to the concentration of the analyteof interest. In one embodiment, a lipid bilayer membrane is depositedacross the orifice and used as a support of biological transmembrane ionchannels for single channel recording. For instance, protein ionchannels, such as α-hemolysin, engineered or chemically modified tointeract with an analyte of interest, are inserted into the bilayermembrane. Binding of the analyte of interest to the protein ion channelsresults in a modulation of ionic current through the nanopore. Incertain embodiments, appropriate molecules (e.g., antigens, singlestranded DNA) are attached to the interior surface of the nanopore toselectively detect proteins and DNA.

Nanopore Membrane

Also provided is a membrane having a thickness, having a first andsecond side, the first side being opposite to the second side, andhaving a nanopore extending through the membrane over the thickness ofthe membrane. A nanopore membrane is exemplified in FIG. 1(C).

In various embodiment of the invention, the membrane may be made ofglass, Si, SiO₂, Si₃N₄, quartz, alumina, nitrides, metals, polymers orother suitable materials. The membrane can be of a pure substance or acomposite, or if necessary, comprises a coating that modifies thesurface of the material. In a particular embodiment, the substrate is asoda lime or lead glass capillary. The thickness of the membrane istypically the smallest dimension of the membrane. The membrane rangestypically from about 10 μm to several hundreds of micrometer inthickness.

The membrane may be configured to include more than one nanopore, or anarray of nanopores. Each individual nanopore may be enclosed in anindividual chamber and such individual chambers may be arranged in anarray format on suitable support structures.

In various embodiments, the nanopore has a first opening and a secondopening. The first opening opens to the first side of the membrane andthe second opening opens to the second side of the membrane. The twoopenings may be of different sizes or shapes. Preferably, the firstopening is smaller than the second opening. In particular, the nanoporeis of a truncated conical shape, wherein the first opening is smallerthe second opening. The radius of the first opening of the nanoporepreferably ranges from about 2 nm to about 500 nm, or larger. Radius ofthe second opening can be about 1 μm to 25 μm. Since the nanoporeextends through the membrane, and connects the first side and the secondside of the membrane, the thickness of the membrane is typically thelength or depth of the nanopore if the thickness of the membrane isuniform across the membrane. The length of the nanopore is preferably 20times of the radius of the first opening of the nanopore. The length ofthe nanopore may range from about 20 μm to about 75 μm. The position ofthe nanopore may be located at any predetermined position on themembrane.

A characteristic of conical-shaped nanopore electrodes andconical-shaped nanopore membranes, which offers great advantage inanalytical sensor measurements, is that the largest mass-transport andionic resistance of the pore is localized at the pore orifice. Thisfeature is a consequence of the combination of (i) the convergent radialflux of molecules and ions from the bulk solution to the disk-shapedorifice and (ii) the divergent radial flux of molecules from the orificeto the electrode. The flux of molecules and ions from the bulk solutionto the pore thus obtains a maximum value at the orifice that may beorders of magnitude larger than the flux at the bottom of the pore. Thisgeometry-based localization of the pore resistance at the orificeenhances applications of the conical-shaped nanopores by providing asmall-volume and high-resistance transduction region at the smallorifice, while the remainder of the wider-region pore provideslow-resistance access to the transduction region.

An additional advantage of the conical pore shape described herein isthat ion and molecule fluxes through conical pores asymptoticallyapproach a constant value when the depth of the cone-shaped pore is ˜50×larger than the radius of the pore orifice, a. The resistance of aconical pore also asymptotically approaches a constant value when thedepth of the cone-shaped pore is ˜50× larger than the radius of the poreorifice, a.

Further provided are methods of preparing a modified or non-modifiednanopore membrane, such a method comprising preparing a modified ornon-modified nanopore electrode including an ISTE as disclosed herein,and removing the ISTE leaving a nanopore in the membrane.

DESCRIPTION OF THE FIGURES

FIG. 1 (A) depicts a nanodisk electrode; FIG. 1 (B) depicts a glassnanopore electrode, and FIG. 1 (C) depicts a glass nanopore membrane.

FIG. 2 (A) depicts a nanopore electrode, FIG. 2 (B) depicts a chemicallymodified nanopore electrode and FIG. 2 (C) depicts the geometry of ananopore electrode.

FIG. 3 is a schematic of a conical shaped nanopore in a thin glassmembrane.

FIG. 4 illustrates procedures of preparing a glass nanopore membrane:(a) preparation of Pt tip, (b) insertion of a Pt ISTE into a glasscapillary, (c) sealing of the Pt ISTE, (d) polishing of the glasscapillary to produce a nanodisk electrode, and (e) etching of the PtISTE to produce a nanopore electrode, and (f) removal of the ISTE toproduce a nanopore membrane.

FIG. 5 (a) is a schematic of a Pt ISTE sealed in a glass membrane. FIG.5 (b) shows an electrical circuit that is able to control the size of ananopore in a glass membrane. FIG. 5 (c) is a photograph of anelectrical circuit device attached to a Pt ISTE sealed in a glassmembrane and a polishing cloth.

FIGS. 6A-6D illustrate chemical modification of the exterior andinterior surface of a nanopore electrode.

FIG. 7. Electron microscopy images of (A) Pt (SEM) and (B) Au (TEM)wires after etching in 6 M NaCN/0.1 M NaOH.

FIG. 8. (A) Schematic of a cell and (B) pulse waveform for preparingultra-sharp Pt tips.

FIG. 9. High-resolution TEM image of a Pt tip following sharpening in0.1 M H₂SO₄.

FIG. 10. (A) Resistance of a glass-sealed Pt disk electrode duringpolishing computed from finite-element simulations. Inserts depict thepotential distribution before and after exposure of the Pt disk. (B) SEMof resulting disk electrode prepared using the finite elementsimulations and high-sensitivity electrical circuit for controlling theradius of the Pt disk

FIG. 11. (A) Circuit diagram of the high-sensitivity electricalcontinuity tester for detecting first exposure of Pt. (B) Circuitdiagram of the high-sensitivity electrical circuit for controlling theradius of the Pt disk. R_(G) is a variable resistor that allowsselection of the disk radius.

FIG. 12. A graph depicting voltammetric response of a Pt disk electrodein CH₃CN containing 5 mM Fc and 0.2 M TBAPF₆ (A) before and after (B)initial Pt tip exposure (˜0.4 nm radius), and (C) after further tipexposure to create a disk electrode.

FIG. 13. Graphs depicting voltammetric responses of Pt nanodiskelectrodes measured in CH₃CN/0.2 M TBAPF₆ in the presence and absence of5.0 mM Fc. (A) 11-nm radius sealed in soda-lime glass and (B) 15 nm-sealin Pb glass (sweep rate=20 mV s⁻¹).

FIG. 14. Graphs depicting voltammetric responses of Au nanodiskelectrodes measured in CH₃CN/0.2 M TBAPF₆ in the presence and absence of5.0 mM Fc. (A) 21-nm radius sealed in soda-lime glass and (B) 49 nm-sealin Pb-doped glass (sweep rate=20 mV s⁻¹).

FIG. 15. Plot of experimentally measured radius of Pt nanodisks vs thepre-selected value. Nanodisks were fabricated using the MOSFET circuitin FIG. 11(B) and finite element simulations shown in FIG. 10. The insetshows a scanning electron micrograph of a Pt nanodisk electrode.

FIG. 16. Graph depicting voltammetric response of an 86-nm-radius Ptnanodisk electrode and the corresponding nanopore electrode in CH₃CNcontaining 5.0 mM Fc and 0.2 M TBAPF₆.

FIG. 17. (A) Voltammetric response of a 23-nm-radius Pt disk electrodein CH₃CN containing 5.0 mM ferrocene and 0.2 M TBAPF₆. (B) AFM image ofthe orifice after the Pt is partially removed from the electrode in part(A). A 23-nm scale bar is shown across the nanopore.

FIG. 18. Optical images during the preparation of a glass nanoporemembrane. (A) Pt sealed in bulk glass; (B) Pt sealed in glass membraneafter polishing glass; (C) glass nanopore membrane after removal of Pt;and (D) schematic of glass nanopore membrane.

FIG. 19. (A) i-V curves for a 32-nm-radius glass nanopore membrane as afunction of KCl concentration (the potential is measured across themembrane; scan rate 100 mV s⁻¹). (B) Voltammetric response of thecorresponding Pt nanodisk in a 10.0 mM Ru(NH₃)₆Cl₃ solution containing 1M KCl (scan rate 20 mV s⁻¹). The pore orifice size, as assessed from theslope of the i-V curve in (A) for 1 M KCl is 31 nm), which is equal,within error, to the value obtained from the Faradic response in (B)(a=32 nm).

DETAILED DESCRIPTION OF THE INVENTION

It is particularly useful to develop a structurally simple and reliablenanopore platform for investigating molecular transport through orificesof nanoscale dimensions. Provided is a surface-modified nanoporeelectrode with a built-in ISTE, the preparation and use thereof. Incontrast to analytical measurements based on pores in free-standingmembranes, the glass nanopore electrode is open to solution though asingle orifice. Advantages of this design include: simplicity andreproducibility of fabrication; a built-in signal transduction element(e.g., a Pt electrode) for monitoring transport through the pore (eithermolecular transport or ion conductance); and mechanical robustness ofthe solid electrode. The device is portable, relatively inexpensive toproduce, and can be readily expanded to an array of nanopore electrodesensors for simultaneous detection of multiple analytes. The deviceconcept can also be incorporated into silicon and other microelectroniclithographically fabricated devices. These analyzers can be used assensors for pharmaceutical industry, homeland security, and militaryapplications.

In the following description, reference is made to the accompanyingdrawings, which show, by way of illustration, several embodiments of theinvention.

FIG. 2 (A) depicts a glass nanopore electrode and FIG. 2 (B) depicts asurface-modified glass nanopore electrode. In FIGS. 2 (A) and (B),nanopore electrode 100 comprises glass substrate 110, Pt wire 120, andnanopore 130. Glass substrate 110 has first surface 112 and secondsurface 124. First surface 112 is also called the exterior surface ofnanopore 130. First surface 112 is modified by first entity 116. Firstentity 116 that was attached to first surface 112 changes the surfaceproperty of first surface 112. The choice of first entity 116 depends onthe surface property desired. Pt wire 120 has first surface 122 andsecond surface 124. First surface 122 is embedded in substrate 110. Ptwire 120 may extend through substrate 110 on the end comprising secondsurface 124. Optionally, Pt wire 120 does not extend through secondsurface 114 of substrate 110 as long as second surface 124 is exposedthrough second surface 124. Nanopore 130 comprises interior surface 132,circular orifice 134, and circular base 122. Orifice 134 opens throughfirst surface 112 of substrate 110. It is to be noted that base 122 ofnanopore 130 is the first surface of Pt wire 120. Base 122 is alsocalled a disk, in this case, a Pt disk. Interior surface 132 is modifiedwith second entity 136. Second entity 136 that is attached to interiorsurface 132 changes the surface property of interior surface 132. Thechoice of second entity 136 depends on the surface property desired.Nanopore 130 is of truncated conical shape with the radius of orifice134 smaller than the radius of base 122. The radius of orifice 134 istypically ranging from 2 nm to 30 μm. The depth, designated as d in FIG.2, is typically ranging from 10 nm to 100 μm.

The geometry of a truncated cone-shaped nanopore electrode, shown inFIGS. 2(A) and (B), is further illustrated in FIG. 2(C). Nanopore 130can be characterized by any three of the following four parameters: thedepth of the pore, d, the radius of the Pt disk (e.g., base 122 in FIG.2(A)) at the bottom of the pore, a_(p), the orifice radius, a, and thehalf-cone angle Θ. The angle Θ is determined by the cone angle of theetched Pt wire before it is sealed in glass, which is readily measuredby optical microscopy to within 1° as described in Zhang et al., Anal.Chem. 2004, 76, 6229-6238. The radius of the orifice, a, is equal to theradius of the exposed Pt disk (e.g., Pt disk in FIG. 1) prior to etchingthe Pt.

FIG. 3 is a cross sectional view of a truncated cone-shaped nanopore ina thin glass membrane. In FIG. 3, nanopore device 100 comprises glasscapillary 110, and nanopore 120. Glass membrane 130 is an integral partof glass capillary 110. Glass membrane 130 has a first side 140 and asecond side 150. Nanopore 120 extends through glass membrane 130, thusforms a channel connecting the first side and the second side of glassmembrane 130. Nanopore 120 has first opening 160 facing the first sideof glass membrane 130, and second opening 170 facing the second side ofglass membrane 130. First opening 160 is smaller than second opening170. Typically, first opening 160 is ranging from 2 nm to 500 nm; andsecond opening is ranging from 5 μm to 25 μm. The thickness of glassmembrane 130, also the length of nanopore 120 in this case, is ˜20-75μm.

General procedures of preparing a glass nanopore membrane areschematically depicted in FIG. 4. Preparations of nanodisk electrodes,nanopore electrodes and nanopore membrane are further illustrated usingExamples herein. The preparation for a nanopore membrane typicallyinvolves four major steps: preparing an ISTE with a cone-shaped tip,sealing the tip of the ISTE in a glass substrate, polishing thesubstrate until the tip is exposed to produce a nanodisk electrode,etching the tip the ISTE to produce a nanopore electrode, and removingthe ISTE to produce a nanopore membrane. In FIG. 4 (a), electrode 280comprises Pt wire 210 which is attached to W rod 260 via Ag paint. Ptwire 210 is electrochemically etched to produce a sharp tip. In FIG. 4(b), electrode 280 with sharpened Pt tip 210 is inserted inside glasscapillary 220. In FIG. 4 (c), glass capillary 220 is sealed to producebottom wall 230 in which Pt tip 210 is embedded. In FIG. 4 (d), sealedbottom wall 230 of capillary 220 is polished until Pt disk 240 isexposed. In FIG. 4 (e), exposed Pt disk 240 is electrochemically etchedto produce a nanopore. In FIG. 4 (f), the Pt wire is removed by gentlypulling the W rod (connected to the Pt) or by electrochemical orchemical etching of the Pt wire.

The size of the orifice of a nanopore (e.g., orifice 134 as shown inFIG. 2(A), or first opening 160 in FIG. 3, can be controlled bycontrolling the size of the exposed disk of an ISTE. To aid polishing anelectrical continuity circuit may used to signal the exposure of themetal during polishing. The circuit can be designed such that a user isalerted at precisely the moment that the disk of the ISTE is firstexposed during polishing. One way to accomplish this is to analyze theelectrical continuity between the ISTE sealed in the substrate and apolishing means such as an electrolyte-wetted polishing cloth, e.g.,analyzing the electrical resistance as function of the thickness of thesubstrate above the tip of the ISTE during polishing. The combinedresistance of the ISTE, the substrate, and the polishing cloth isreferred to herein as the resistance of the polishing circuit. The totalresistance between the ISTE embedded in the substrate and the substratesurface in contact with the polishing cloth may be computed using finiteelement simulations, which enables estimation of the size of the exposeddisk during polishing. The method of using finite element simulations toaid controlling of the disk size is further demonstrated in theaccompanying example.

FIG. 5 depicts an electrical feedback circuit that can be used tocontrol the size of a nanopore. As shown in FIG. 5 (c), electrode 410comprises a Pt tip with an extended W rod, wherein the Pt tip is sealedin glass capillary 420. Glass capillary 420 is initially sanded using400, 800, 1200 grit papers until ˜0.5 μm of glass remained above the Pttip. Glass capillary 420 is then polished on felt polishing paper wettedwith an aqueous slurry of polishing powder containing an electrolyte,e.g., 0.1 M KCl. An electrical continuity measurement using device 440,which may be based on a MOSFET “on/off” switching circuit, is made todetermine when the DC resistance between the W rod extending from thetop of the capillary and the wetted polishing cloth decreased below 1-2GΩ, signaling exposure of the Pt tip. Polishing is immediately ceased atthis point. The 1-2 GΩ resistance has been empirically determined toyield exposed Pt disks (e.g., Pt disk 240 in FIG. 4) withelectrochemically-determined radii in the range 2 to 30 nm. The signalresistance and circuit may be modified to produce exposed Pt disks ofdifferent radii, and thus provide control of the size of the orifices.

An example of chemical modification of the exterior and interior surfaceof a nanopore electrode is depicted in FIG. 6. In FIG. 6( a), Pt wire512 with a conical tip is sealed in glass substrate 510. Surface 514 ispolished to produce exterior surface 516 and to expose Pt disk 518, asshown in FIG. 6 (b). Exterior surface 516 is first protected by covalentattachment of an alkane silane that possesses a terminal “inert”functionality. The purpose of inert terminus of this monolayer is toprevent binding or specific interaction of molecules and analytes withthe exterior surface. Exposed Pt disk 518 is then electrochemicallyetched to produce pore 520 which results in Pt disk 522 at the porebase. Pt disk 522 serves as an electrical signal transducer forelectrochemical or conductivity measurements. Interior surface 524 ofthe glass pore is then modified to introduce binding sites or chemicalfunctionality that responds to external stimuli. For example, an —NH2terminating silane may be attached to interior surface 524, as the aminegroup is a convenient starting point for coupling to analyte-specificligands.

Some embodiments of the invention are disclosed in Zhang, Anal Chem.,2004, Zhang, Anal Chem., 2006; Zhang, JPC, 2006, Wang, JACS 2006, White,Langmuir, 2006.

The invention is further described with the aid of the followingillustrative Examples.

EXAMPLES

Fabrication of glass nanodisk electrodes, glass nanopore electrodes, andglass nanopore membrane.

Electrochemical Etching of Au and Pt Tips A 2-cm length of Pt or Au wireis connected to a W rod using Ag conductive epoxy (DuPont). The Pt/W orAu/W ensemble is heated in an oven at 120° C. for about 15 minutes todry the Ag epoxy. The end of the Au or Pt wire is electrochemicallyetched to a sharp point in 6 M NaCN/0.1 M.NaOH solution followingstandard methods reported elsewhere ((a) Melmed, A. J. J. Vac. Sci.Technol. B 1991, 9, 601. (b) Melmed, A. J.; Carroll, J. J. J. Vac. Sci.Technol. A 1984, 2, 1388). Briefly, a 100-300 Hz AC voltage (˜4 Vamplitude) is applied between the Pt or Au wire and a large area Ptelectrode using an Agilent 33220A function/arbitrary generator. Bubblesformed at the metal/solution interface during electrochemical etching;the applied voltage is removed immediately upon cessation of bubblingand the sharpened wire is washed with H₂O. Pt tips were furthersharpened, as described herein below, using a custom-designed waveformgenerator.

FIG. 7 shows electron micrographs of Pt and Au tips after etching in 6 MNaCN/0.1 M NaOH. Although similar conditions are used (e.g., ˜180 Hz,3.6 V for Pt, 4.5 V for Au), the etching of Au wires yields tips withsignificantly smaller radii of curvature (<10 nm) than Pt (˜30 nm).

Systematic studies revealed several important correlations. First,larger diameter metal wires result in higher diameter cone-angles at thetip. For example, etching a 25-μm-diameter Pt wire yields tips withhalf-cone angles of 8.5±1°, while etching a 100-μm-diameter Pt wireyields tips with half-cone angles of 14±1°. The ability to control thecone angle of the tip is of practical utility, as the transportresistance of the glass nanopores is sensitive to this parameter.Second, the surface roughness of the etched metal tips, especially forPt, is very dependent on the frequency of the applied AC voltage. Higherfrequencies yield significantly smoother surfaces. However, thefrequency of the etching voltage are preferably less than 1000 Hz inorder to produce sharp tips. Empirically a frequency-range of 110-300 Hzyields tips that are satisfactory for producing nanodisks.

Electrochemical Sharpening of Pt Tips Nanodisk electrodes with radiibetween 30 and 100 nm can be fabricated using Pt tips sharpened asdescribed above. To fabricate even smaller Pt electrodes, the proceduredescribed by Libioulle et al. (Libioulle, L.; Houbion, Y.; Gilles, J.-M.Rev. Sci. Instrum. 1995, 66, 97) for sharpening Pt tips for use in STMwas adopted, with a few modifications. FIG. 8A shows a U-shapedelectrochemical cell that is employed for this purpose. This cell has alarge reservoir on the left-hand side that is used to position theair-solution (0.1 M H₂SO₄) interface in the horizontal glass tube atright. The end of an etched Pt tip is inserted orthogonally across themeniscus of the 0.1 M H₂SO₄ within the horizontal glass tube. A pulsedvoltage waveform is then applied to electrochemically sharpen the tipfurther. A 4 kHz, 15 V, 16 μs pulse waveform was applied for 1 s with ahomebuilt waveform generator, followed by a DC potential of −1.1 V for10 s to remove any PtO_(x). The voltage program as a function of time isrepresented in FIG. 8B. Repetition of the program was carried out threetimes in succession to obtain sufficiently sharp tips for electrodefabrication.

FIG. 9 shows a TEM image of a typical Pt tip sharpened by the procedureoutlined herein above. The radius of curvature of the tip is ca. 2 nmand the surface appears free from oxide deposits. TEM characterizationindicates that the majority of Pt tips processed using the program inFIG. 8B have radii <10 nm.

Sealing Pt and Au Tips in Glass The sharpened end of the Pt or Au wireis inserted into a glass capillary, leaving ˜3 mm between the tip andthe end of the glass tube. The wire is then sealed into glass tube byslowly softening the capillary in a H₂—O₂ flame. An optical microscopeis used to frequently check the quality of the seal during this process(e.g., to ensure that no air bubbles became trapped near the metal tip).After obtaining an acceptable seal, the top of the W rod is secured tothe glass capillary with epoxy (Dexter). Rough polishing to remove alarge portion of the glass (e.g., by leaving ˜100 μm between the metaltip and the outside edge of the capillary) is accomplished using finesand paper or emery cloth. Final polishing to expose the Pt or Au diskis performed using a wetted Buehler MICROCLOTH™ polishing pad mounted ona green glass plate with the aid of an electrical continuity tester asdescribed herein below.

Two primary conditions must be met in order to seal the metal into aglass capillary without destroying the ultra-sharp Pt and Au tips.First, the thermal expansion coefficient of the glass should be equal orgreater than that of the metal to prevent crevice formation uponcooling. Secondly, the sealing temperature must be much lower than themelting point of the metal in order to avoid changes in tip shape. Thus,the softening temperature of the glass should be significantly lowerthan the melting point of the metal.

Table 1 lists the melting points and linear expansion coefficients ofthe metals and glasses used in this study. The melting point of Pt(˜1770° C.) is ˜1000° C. higher than the softening point of either sodalime or Pb-doped glass, and the expansion coefficients of Pt and bothglass types are comparable. These conditions indicate that Pt is wellsuited for sealing in either type of capillary. Although Au has asignificantly higher thermal expansion coefficient than either soda limeor lead glass, and a melting point (˜1060° C.) that is only 300-400° C.higher than the glass softening points, we have successfully sealed Auin Pb-doped glass capillaries (as judged from the voltammetricresponse).

TABLE 1 Melting/Softening Points and Expansions Coefficients of Pt, Au,and Glasses. Linear Thermal Expansion Electrical Melting/SofteningCoefficient (25° C.) conductivity Material Point ° C. (×10⁶) K⁻¹ (Ωm)⁻¹Platinum 1769 9 9.3 × 10⁶ Gold 1064 14 4.4 × 10⁷ Soda Lime ~700 9.3~10⁻¹⁰ Glass Pb-doped ~600 9.5 ~10⁻¹⁴ Glass

Polishing of Glass Electrode To aid hand polishing, a high-inputimpedance (MOSFET)-based electrical continuity circuit is used to signalthe exposure of the metal during polishing. The electrical continuitybetween the Pt or Au wire sealed in glass and the felt polishing cloth(wetted with a KCl solution and connected to the external circuit with ametal clip) is measured. The successful implementation of this strategyhinges on designing the circuit such that the user is alerted atprecisely the moment that the metal is first exposed during polishing.One way to accomplish this is to utilize an analysis of the electricalresistance as function of the thickness of the glass above the tipduring polishing. The combined resistance of the Pt wire, the glass, andthe electrolyte-wetted polishing cloth is referred to here as theresistance of the polishing circuit.

The total resistance between the Pt wire embedded in the glass and theflat glass surface in contact with the polishing cloth is computed usingfinite element simulations. The electrical conductivity of glass is setat 10⁻¹⁰ (ohm·m)⁻¹, typical of soda lime glass (Table 1) andapproximately 17 orders of magnitude lower than Pt. Prior to Ptexposure, the glass layer between the metal and the polishing cloth isby far the dominant resistance (the resistance of the solution can beignored). Upon exposure of the tip, the spreading resistance at thenanodisk/electrolyte interface becomes controlling, and computed usingthe equation: (Bard, A. J.; Faulkner, L. R. Electrochemical Methods:Fundamentals and Applications; 2nd Edition, 2001)R=(4κa)⁻¹  (1)where κ is the conductivity of the 0.02 M KCl solution (κ˜0.14 (ohmm)⁻¹) on the polishing cloth and a is the radius of the metal disk.Other KCl concentrations and different electrolytes can be employed forpolishing using the electrical feedback circuit. Since the voltage dropin the electrolyte occurs over a very small distance (˜10a), it is notnecessary to precisely model the electrolyte layer geometry on thepolishing cloth.

FIG. 10 shows a plot of the logarithm of the polishing circuitresistance (log R (Ω) as a function of the polishing depth, L (nm). Thevalue of L=0 corresponds to the glass surface aligned with the end ofthe metal tip. Thus, negative values of L correspond to the thickness ofthe glass layer before the tip is exposed, while positive valuescorrespond the thickness of the glass removed following tip exposure. Ageometric analysis can be used to convert L values to the exposed diskradius (a; selected a values are shown in FIG. 10). Thus, in principle,measurement of the polishing circuit resistance enables estimation ofthe size of the metal disk during polishing.

To obtain real-time feedback, a MOSFET circuit as a DC continuity testeris used to determine the moment during polishing when the metal tipbecomes exposed. From the simulations in FIG. 10A we can definecontinuity as a decrease in R from ˜10¹⁸Ω (immeasurable) to ˜10⁹Ω. Anotable progression is R=8.9 GΩ for a=0.1 nm, 4.5 GΩ when a=0.2 nm, and1.8 GΩ at a=0.5 nm. In the circuit described herein below, electricalcontinuity is signaled when R=˜2 GΩ, corresponding to a metal nanodiskwith a ˜0.5 nm (exposure of ˜10 metal atoms of radius 0.15 nm in acircular disk would meet this criteria). FIG. 10B shows a SEM of a Ptdisk exposed at the surface of glass produced by this method.

A circuit diagram of the continuity tester is shown in FIG. 11A. Aresistance of less than ˜2 G ohms is indicated by an audio signal and/orlighted LED. Although a more sensitive tester could be developed, suchhigh sensitivity devices are susceptible to false readings due to verysmall leakage currents through surface contamination of the test leads,and through conduction or tunneling across the nanometer-thin glasslayer just prior to exposing the tip.

The signaling mechanism consists of a solid-state beeper and an LED,which are both connected between the +9V battery terminal and the drainlead of a VL0300L MOSFET transistor. The source lead of this transistoris connected to the negative terminal of the battery. The test leads areconnected such that the transistor acts as a switch in the circuit; whena complete circuit is created by a resistance of less than ˜2 GΩ betweenthe test leads, the beeper will sound and the LED will turn on. This isaccomplished by attaching one test lead directly to the +9V batteryterminal and the other to the transistor gate via a 500 MΩ resistor thatis wired in series to the negative battery terminal. If there issufficient continuity through the test leads, a 1.5 V drop across the500 MΩ resistor will activate the transistor. The enhancement mode FETis normally off, and therefore the beeper and LED are normally off.However, ˜1.5V at the gate of the FET activates the transistor andtherefore sets off the beeper and the LED.

In operation, one test lead is connected to the W rod that contacts thePt or Au wire embedded in the glass capillary, and the other lead isbathed in the solution on the polishing cloth. When enough glass hasbeen polished to just barely expose the Pt/Au tip, sufficient currentwill flow through the probe, causing a voltage drop across the 500 MΩgate resistor, which in-turn activates the transistor.

Upon first exposure of the metal, an intermittent audio or LED signaloccurs, which is possibly due to capacitive currents. An additional fewseconds of polishing results in a continuous signal that is probablyassociated with oxidation or reduction of H₂O or other redox activeconstituents of the electrolyte (Cl⁻, O₂) that wet the polishing cloth.

The polishing circuit of FIG. 11A can be modified by replacing the 500MW resistor with a variable resistor R_(G) as shown in FIG. 11B. Thisresults in an electrical, optical or audio signal to the polisher that adisk electrode of size, determined by the calculations in FIG. 10A andthe value of R_(G), has been produced. Polishing is immediately ceasedupon indication of the desired disk size either by the LED or audiosignal.

The radius of the exposed metal disk can be determined by several means,including: steady-state voltammetry, atomic force microscopy,conductance measurements (of the pore after removal of the metal), andelectron microscopy. Nanodisk radii are measured by steady-statevoltammetry, which is by far the least intensive method. Representativesamples are also characterized by an additional method to establishcorrelations with the voltammetric measurements, ensuring mutualvalidity. In voltammetry, the radius of the nanodisk is assessed usingthe steady-state limiting current, i_(d), for the oxidation of a solubleredox species through the equation (Saito, Y. Rev. Polarog. (Japan)1968, 15, 177.)i _(d)=4nFDCa  (2)where n is the electron stoichiometry, F is Faraday's constant, and Dand C* are the diffusion coefficient and bulk concentration of the redoxmolecule, respectively. Values of a were determined by measuring i_(d)for the oxidation of 5.0 mM ferrocene (Fc, D=1×10⁵ cm² s⁻¹) inacetonitrile (supporting electrolyte 0.2 M TBAPF₆).

FIG. 12 shows the voltammetric response of a Pt disk electrode in 5.0 mMFc/0.2 M TBAPF₆ solution before tip exposure (A), immediately after thecontinuity tester signals exposure with intermittent beeping (B), andafter sonicating the nanodisk in ethanol for ˜20 s (C). Prior toexposure of the metal tip, there is a small charging current (˜0.5 pA,FIG. 12A) that corresponds, at least partially, to the stray capacitanceof the instrument. This background capacitance is observed whether ornot the metal nanodisk is exposed, and appears even when the capillaryand metal wire is removed entirely from the solution. The voltammetriccurve in FIG. 12B shows an increase in current that is nearlyexponential beginning near E^(0′) (the thermodynamic redox potential).This current does not reach a diffusion-limited plateau and may arisefrom any combination of either (1) conductance or tunneling across avery thin glass layer (<1 nm) that remains above the Pt tip, or (2)kinetically controlled oxidation of Fc at a sub-nanometer-radiusnanodisk. FIG. 12C shows that a voltammetric response exhibiting awell-defined diffusion-limited current plateau is obtained after a briefsonication of the electrode (5 s). Sonication is believed to remove anyremaining glass at the electrode surface, albeit in an uncontrolledfashion. The voltammetric response after sonication corresponds to aradius ˜4 mm.

Continuing polishing briefly beyond the intermittent beeping stage(until a continuous audio alert is obtained) results in electrodes thatgive well-defined voltammetry. i-V curves in electrolyte only and 5.0 mMFc solutions for two different Pt electrodes is presented in FIG. 13.These electrodes were prepared using soda lime glass (a=11 nm, FIG. 13A)and Pb-doped glass (a=15 nm, FIG. 13B). Similar voltammetric responseswere obtained for glass-shrouded nanodisks prepared using sharpened Auwires, FIGS. 14A and 14B. Using the circuits in FIG. 11, we find that Ptand Au nanodisks with radii ranging between 2 and 25 μm can be routinelyprepared, the majority of which exhibit nearly ideal voltammetricbehavior (similar to the examples presented herein).

Close examination of FIG. 13 reveals a difference in the voltammetricresponse of the two Pt electrodes prepared using soda lime and Pb-dopedglass capillaries. The i-V response of the Pt electrode shrouded in sodalime glass exhibits a significantly larger linear background slope (75pA/V) than that of the Pb-doped glass (immeasurably small). This slopeis not due to a poor glass/metal seal, but instead reflects thesteady-state flux of Na⁺ within the bulk soda lime glass. Thissupposition is supported by experiments in which a Pt wire is sealedentirely in soda lime glass (no polishing). The slopes of the i-V curvesof these electrodes at any time during polishing, but before exposure,are indistinguishable. The conduction process due to the transport ofNa+ within the glass appears superimposed on the voltammetric responsefor Fc oxidation. For example, in FIG. 13A the i-V curve for the sealedPt shows a larger hysteresis than the corresponding i-V curve in FIG.13B, for an electrode sealed in Pb-doped glass. This effect is readilydetectable due to the high current sensitivity used in recording thedata. The reproducible absence of an ohmic background when usingPb-doped capillaries is consistent with the much lower ionicconductivity of Pb-doped glass relative to soda lime glass (see Table1).

As previously discussed, electron microscopy has been used to measurethe radii of our Pt nanodisk electrodes (as well as nanopore electrodessynthesized from nanodisks, see below). In general, we find goodagreement between SEM-determined radii and voltammetric results. AFMimaging and conductivity measurements of nanopore electrode orificesprepared from the nanodisk electrodes yield radii in excellent agreementwith values from voltammetric measurements.

FIG. 15 shows a plot measured Pt disk radii vs values pre-selected byadjusting the resistance R_(G) in the circuit of FIG. 11B. The datademonstrate that the size of the disk can be controlled using thecircuit of FIG. 11B in conjunction with the calculations shown in FIG.10A.

Glass Nanopore Electrodes The glass nanopore electrode (see FIG. 1B) isfabricated by etching the Pt or Au nanodisk electrode in a 20% CaCl₂solution, using a ˜5 V amplitude AC voltage at a frequency of 60 Hz. Theremoval of Pt during etching creates a conical shaped pore, the depth ofwhich is controlled by varying the etching time. The essentialdifference in the behavior of a Pt nanopore electrode with respect to ananodisk electrode is a notable decrease in the diffusion-limitedcurrent. This reduction is a consequence of the larger mass-transferresistance of the pore, and therefore scales with pore size. FIG. 16shows the voltammetric responses of a 86-nm radius Pt disk electrode andthe corresponding glass nanopore electrode in 5.0 mM Fc. This particularnanopore resulted from etching the Pt to a depth of ˜250 nm, asdetermined from the dependence of the limiting current on the ratiobetween the pore depth and the radius of the orifice (d/a) (Bo Zhang,Yanhui Zhang, and Henry S. White, The Steady-State Voltammetric Responseof the Nanopore Electrode, Anal. Chem. 78, 477-483 (2006)).

The ˜65% decrease in limiting current upon formation of the pore, FIG.16, is consistent with published numerical simulations (Anal. Chem. 78,477-483 (2006)). FIG. 17 shows (A) the voltammetric response of a Ptnanodisk electrode that is used to prepare a nanopore electrode, and (B)an AFM image of the orifice of a nanopore electrode that resulted frometching this nanodisk electrode, using the procedure outlined above. ThePt nanodisk radius is determined to be 23 nm from measurement of thediffusion limited current (FIG. 15A). A 23-nm scale bar is shown on theAFM image, illustrating the voltammetric measurement is in excellentagreement with the AFM image. This observation is consistent with SEMstudies of larger pores, where the size of the nanopore andcorresponding nanodisk electrode are similar. These results indicatethat the voltammetric response yields accurate values of metal diskradii, and that electrochemical etching of the Pt in CaCl₂ does notremove a significant amount of glass from the walls of the resultingnanopore.

Glass Nanopore Membranes The sealed metal wire can be removed entirelyfrom the glass by a combined etching and mechanical process to make aglass membrane containing an individual conical shape nanopore. Sealingvery short lengths (25-50 μm) of the sharpened end of a Pt wire isaccomplished using a specialized procedure. First, the tip is positionedat the middle of the glass capillary to avoid touching of the glasswalls while the glass is being heated in the H₂ torch. Initially, the Ptis positioned >0.5 cm from the end of the glass capillary while the endof the capillary is heated. As the capillary softens and collapses, theinterior surface becomes very flat. At this point, the glass capillaryis removed from the flame and the Pt tip is positioned as close aspossible toward the sealed end of the capillary, taking care to avoidphysical touching of the glass surface by monitoring progress with anoptical microscope. The capillary is them placed back into the lower,cooler part of the flame to continue softening the glass with constantvisual inspection of the interior flat surface. As the glass continuesto soften in the flame, it eventually contacts the sharp Pt tip. Thiscontact is observed by eye (with considerable practice) in real time bythe sudden appearance of a spot at the point of contact. The capillary,with Pt tip sealed at the end, is immediately removed from the flame andallowed to cool.

The capillary is polished as described above using the electricalcircuits, FIG. 10, to create a Pt nanodisk, the size of which can becharacterized at this intermediate point. The Pt is thenelectrochemically etched in CaCl₂ to remove as much of the Pt wire aspossible from the glass. The remaining Pt can readily be removed fromthe glass at this point by gently twisting the W wire attached to the Ptinside the capillary. FIG. 18 shows cross-sectional optical images of:(A) Pt tip sealed in glass; (B) the polished Pt nanodisk electrode; (C)the glass nanopore membrane after the tip is removed; and (D) aschematic drawing of the glass nanopore membrane showing the dimensions.

The radius of the small orifice of a glass nanopore membrane can becomputed from the resistance of the pore measured in a solution of knownionic conductivity. The pore resistance is obtained from the slope ofohmic i-V curves recorded by varying the potential between two Ag/AgClelectrodes positioned on opposite sides: of the membrane. Therelationship between the membrane resistance, R_(p), and the smallorifice radius, a, is given by: (Ryan J. White, Bo Zhang, Susan Daniel,John Tang, Eric N. Ervin, Paul S. Cremer, and Henry S. White, “IonicConductivity of the Aqueous Layer Separating a Lipid Bilayer Membraneand a Glass Support,” Langmuir, 22, 10777-10783 (2006)).

$\begin{matrix}{R_{p} = {\frac{1}{{\kappa\; a}\;}\left( {\frac{1}{4} + \frac{1}{\pi\;\tan\;\theta}} \right)}} & (3)\end{matrix}$where R_(p) is the resistance, κ is the conductivity of the solution,and θ is the half-cone angle. The latter is equal to the half-cone angleof the Pt wire before it is sealed, which is measured by opticalmicroscopy.

FIG. 19B shows the voltammetric response of a Pt nanodisk electrode in a10 mM Ru(NH₃)₆Cl₃ solution containing 0.2 M KCl. The radius of the Ptdisk is calculated from the diffusion-limited steady-state current to be32 nm. FIG. 19A shows the voltammetric response of the correspondingglass nanopore membrane in KCl solutions of varying concentration. Thei-V response in 1 M KCl is found to be ohmic, with a slope correspondingto a small orifice radius of ˜31 nm, in good agreement to the valuecalculated from the electrochemical response. The i-V response in KCl oflow concentrations displays current rectification effects, similar tothat reported by Wei et al. for current flow through tapered glassnanopipettes (Wei, C.; Bard, A. J.; Feldberg, S. W. Anal. Chem. 1997,69, 4627.) Current rectification in conical nanopores has been recentlyreviewed by Siwy (Siwy, Z. S. Adv. Funct. Mater. 2006, 16, 735).

While this invention has been described in certain embodiments, thepresent invention can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of the invention using its generalprinciples. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and which fallwithin the limits of the appended claims.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein. The references discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention.

1. A nanopore membrane, comprising: a membrane having a thickness, witha first, exterior, side and a second, interior, side, with the firstside being opposite the second side, wherein the membrane is made from amaterial selected from the group consisting of glass, Si, SiO₂, Si₃N₄,quartz, alumina, nitrides, metals and polymers; and a nanopore extendingthrough the membrane, thus forming a channel connecting from the firstside to the second side of the membrane, wherein the nanopore has afirst opening that opens to the first side of the membrane, a secondopening that opens to the second side of the membrane and a depth,wherein the surface properties of at least one of: the first side of themembrane; the second side of the membrane; and the channel connectingthe first side to the second side of the membrane are modified to changeat least one of an electrical charge density, hydrophobicity andhydrophilicity property thereof, with the first side of the membranebeing modified with a first entity, and the channel connecting the firstside to the second side of the membrane being modified with a secondentity which is different from the first entity.
 2. The nanoporemembrane of claim 1, wherein the nanopore is of a truncated conicalshape and wherein a radius of the first opening of the nanopore issmaller than a radius of the second opening of the nanopore.
 3. Thenanopore membrane of claim 1, wherein the radius of the first opening ofthe nanopore ranges from about 2 nm to 500 nm.
 4. The nanopore membraneof claim 1, wherein the radius of the first opening of the nanopore islarger than 500 nm.
 5. The nanopore membrane of claim 1, wherein thefirst entity is a reactive silane with an inert terminus, and the secondentity is a saline that comprises a first functional group able to reactwith the membrane and a second functional group able to react with asensor element able to selectively bind a target analyte.
 6. Thenanopore membrane of claim 1, wherein the first entity isCl(Me)₂Si(CH₂)₃CN, and the second entity is EtO(Me)₂Si(CH₂)₃NH₂.
 7. Ananopore membrane comprising: a membrane having a thickness, with afirst, exterior, side and a second, interior, side, with the first sidebeing opposite the second side, wherein the membrane is made from amaterial selected from the group consisting of glass, Si, SiO₂, Si₃N₄,quartz, alumina, nitrides, metals and polymers; and a nanopore extendingthrough the membrane, thus forming a channel connecting the first sideto the second side of the membrane, wherein the nanopore has a firstopening that opens to the first side of the membrane, a second openingthat opens to the second side of the membrane and a depth; wherein themembrane is a bottom wall of a capillary which is sealed to produce thebottom wall.
 8. The nanopore membrane of claim 7, wherein the surfaceproperties of at least one of: the first side of the membrane; thesecond side of the membrane; and the channel connecting the first sideto the second side of the membrane are modified to change at least oneof an electrical charge density, hydrophobicity and hydrophilicityproperty thereof.
 9. The nanopore membrane of claim 7, wherein thecapillary is made from glass or quartz.
 10. The nanopore membrane ofclaim 7, wherein the nanopore is of a truncated conical shape andwherein a radius of the first opening of the nanopore is smaller than aradius of the second opening of the nanopore.
 11. The nanopore membraneof claim 7, wherein the radius of the first opening of the nanoporeranges from about 2 nm to 500 nm.
 12. The nanopore membrane of claim 7,wherein the radius of the first opening of the nanopore is larger than500 nm.
 13. The nanopore membrane of claim 7, wherein the first side ofthe membrane is modified with a first entity, and the channel connectingthe first side to the second side of the membrane is modified with asecond entity.
 14. The nanopore membrane of claim 13, wherein the firstentity is a reactive silane with an inert terminus, and the secondentity is a saline that comprises a first functional group able to reactwith the membrane and a second functional group able to react with asensor element able to selectively bind a target analyte.
 15. Thenanopore membrane of claim 14, wherein the first entity isCl(Me)₂Si(CH₂)₃CN, and the second entity is EtO(Me)₂Si(CH₂)₃NH₂.
 16. Thenanopore membrane of claim 13, wherein the first entity and the secondentity are the same.